Loss of cell-cell interactions, degradation of the basement membrane and invasion of the underlying stromal tissue by malignant epithelial cells are key events in the progression of a primary breast tumor to local and distant metastases. There is increasing evidence that proteolytic enzymes, or proteases, are involved in tumor progression. In recent years several endosomal/lysosomal cysteine proteases have been cloned and shown to be overexpressed in cancer tissues. However, despite the enormous potential for development of novel therapeutics, we still understand little about the regulation of their proteolytic activity. Cystatins such as cystatin M are potent endogenous protein inhibitors of endosomal/lysosomal cysteine proteases. Cystatin M is expressed in primary human breast cancer but is downregulated in metastatic breast tissue and many cancer cell lines and could therefore represent a novel metastasis suppressor gene. The long-term objective of our studies is to define the critical steps controlled by cystatin M during tumor growth, invasion and metastasis. Our working hypothesis is that cystatin M maintains primary breast tumors in a dormant and non-invasive phase. Tumor progression in vivo is complex in nature. We will therefore begin our work with established in vitro models to determine: 1. whether cystatin M is able to inhibit tumor cell-mediated matrix degradation and invasion. The first specific aim will be addressed using recombinant cystatin M as well as engineering human breast cancer cell lines with altered expression of cystatin M. These in vitro studies should greatly help the principal investigator in designing and performing studies for specific aim 2 that will analyze the suppressing function(s) of cystatin M on primary tumor growth, invasion and metastasis in SCID mice. The new insights that should be generated by the overall research of this project may lead to the design of novel drugs for the treatment of primary invasive human breast cancers and/or their metastases.